Tumor necrosis factor-alpha (TNF) is a cytokine which can cause cell death. The goal of our laboratory is to define the intracellular pathway which leads to TNF-induced lysis and to determine how that pathway is regulated. The TNF-induced lytic response is accompanied by the activation of a cellular phospholipase. Results from our laboratory suggest that by understanding the mechanisms which regulate the activity of this enzyme we will greatly enhance our understanding of the underlying causes of susceptibility and resistance to TNF. The experiments we will perform will utilize the cell line C3HA since this cell line displays a phenotype which is representative of most normal cell types, it is resistant to TNF but can be sensitized with inhibitors of transcription or translation (ITT). Our first aim will be to identify the phospholipase which is activated in C3HA cells following treatment with TNF and ITT which, based on our preliminary experiments, is likely to be the high molecular weight cytoplasmic form of PLA2 known as cPLA2. Our second aim will be to define the signals which are necessary for the activation of this enzyme by TNF and ITT. Ca++, phosphorylation, and proteolysis are the signals we will investigate. Our third aim will be to determine what is different about cells which are susceptible to TNF which permits TNF, on its own, in the absence of ITT, to induce the activity of this phospholipase. We will examine the phospholipase itself, and the signals which control its activity in cells expressing the E1A gene product and tumor-derived cell lines which are susceptible to TNF. Finally, we will ask how the adenovirus E3 region 14.7 kDa protein, heat and sodium arsenite can prevent the TNF-induced activation of this enzyme. Again, experiments have been planned to examine both the enzyme and the signals which are necessary for its activation.